multi-drug resistant escherichia coli in diarrhoeagenic
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Technological University Dublin Technological University Dublin
ARROW@TU Dublin ARROW@TU Dublin
Articles School of Food Science and Environmental Health
2018
Multi-drug resistant Escherichia coli in diarrhoeagenic foals: Multi-drug resistant Escherichia coli in diarrhoeagenic foals:
Pulsotyping, phylotyping, serotyping, antibiotic resistance and Pulsotyping, phylotyping, serotyping, antibiotic resistance and
virulence profiling virulence profiling
C.A. Kennedy
Ciara Walsh
M. Karczmarczyk
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Authors Authors C.A. Kennedy, Ciara Walsh, M. Karczmarczyk, S. O'Brien, N. Akasheh, M. Quirke, S. Farrell-Ward, T. Buckley, U. Fogherty, K. Kavanagh, C.T. Parker, T. Sweeney, and S. Fanning
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VETMIC-2018-404 (revised, version 1)- 2
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Multi-drug resistant Escherichia coli in diarrhoeagenic foals: pulsotyping, 4
phylotyping, serotyping, antibiotic resistance and virulence profiling. 5
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C.A. Kennedya, C. Walshb,c, M. Karczmarczykc, S. O’Brienc,N. Akashehd, M. Quirkeb, 8
S. Farrell-Wardc, T. Buckleye, U. Foghertye, K. Kavanaghe, C.T. Parkerf, T. Sweeneya 9
and S. Fanningc. 10
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aUCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, 13
Ireland, bSchool of Food Sc. and Environmental Health, Cathal Brugha Street, DIT, 14
Dublin D01 HV58, Ireland, cUCD-Centre for Food Safety, School of Public Health, 15
Physiotherapy & Population Science, University College Dublin, Belfield, Dublin D04 16
N2E5, Ireland, dMedical Directorate, St. James’s Hospital, Dublin 8, Ireland, eIrish 17
Equine Centre, Johnstown, Naas, Co. Kildare, W91 RH93, Ireland and fProduce 18
Safety and Microbiology Research Unit, Agricultural Research Service, U.S. 19
Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA. 20
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Corresponding author: Professor Séamus Fanning, 22
UCD-Centre for Food Safety, 23
School of Public Health, Physiotherapy & Sports Science, 24
University College Dublin, Belfield, Dublin D04 N2E5, 25
Ireland. 26
Tel.: (+353) 1 716 2869 27
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© 2018 published by Elsevier. This manuscript is made available under the Elsevier user licensehttps://www.elsevier.com/open-access/userlicense/1.0/
Version of Record: https://www.sciencedirect.com/science/article/pii/S0378113518304310Manuscript_bab5ed40483ef6b85dd58c223e2723cf
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Abstract 31
Extraintestinal pathogenic E. coli (ExPEC) possess the ability to cause extraintestinal 32
infections such as urinary tract infections, neonatal meningitis and sepsis. While 33
information is readily available describing pathogenic E. coli populations in food-34
producing animals, studies in companion/sports animals such as horses are limited. 35
In addition, many antimicrobial agents used in the treatment of equine infections are 36
also utilised in human medicine, potentially contributing to the spread of antibiotic 37
resistance determinants among pathogenic strains. The aim of this study was to 38
phenotypically and genotypically characterise the multidrug resistance and virulence 39
associated with 83 equine E. coli isolates recovered from foals with diarrhoeal 40
disease. Serotyping was performed by both PCR and sequencing. Antibiotic 41
resistance was assessed by disc diffusion. Phylogenetic groups, virulence genes, 42
antibiotic resistance genes and integrons were determined by PCR. Thirty-nine 43
(46%) of the isolates were classified as ExPEC and hence considered to be 44
potentially pathogenic to humans and animals. Identified serogroups O1, O19a, O40, 45
O101 and O153 are among previously reported human clinical ExPEC isolates. Over 46
a quarter of the E. coli were assigned to pathogenic phylogroups B2 (6%) and D 47
(23%). Class 1 and class 2 integrons were detected in 85% of E. coli, revealing their 48
potential to transfer MDR to other pathogenic and non-pathogenic bacteria. With 49
65% of potentially pathogenic isolates harbouring one or more TEM, SHV and CTX-50
M-2 group β-lactamases, in addition to the high levels of resistance to 51
fluoroquinolones observed, our findings signal the need for increased attention to 52
companion/sport animal reservoirs as public health threats. 53
54
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Keywords: Escherichia coli, diarrhoeagenic foals, serotyping, antibiotic resistance, 55
adherence and invasion PFGE, ExPEC, ESBL 56
57
Introduction 58
Pathogenic Escherichia coli infection is a public health challenge and a continuous 59
source of morbidity/mortality (Croxen et al., 2013). Pathogenic E. coli can be split 60
into two categories: intestinal and extraintestinal pathogenic E. coli. Intestinal 61
pathogenic E. coli cause diarrhoea by expressing virulence genes that produce 62
enterotoxins, facilitate attachment and effacement, and/or invasion of the intestinal 63
mucosa. Extraintestinal pathogenic E. coli (ExPEC) possess the ability to cause 64
extraintestinal infections such as urinary tract infections (UTIs), neonatal meningitis 65
and sepsis (Russo and Johnson, 2003). Sources of human infection include direct or 66
indirect contact with animals that carry pathogenic E. coli, and from exposure to 67
animal faeces. Recent outbreaks in public settings, such as farms, fairs and petting 68
zoos, have highlighted the public health impact of this route of transmission (Byrne et 69
al., 2015; Murray et al., 2017). While information is readily available describing 70
pathogenic E. coli populations in food-producing animals (Lenahan et al., 2007; 71
Lenahan et al., 2009; Kennedy et al., 2017), studies in companion/sports animals 72
such as horses are limited. 73
One such study, focusing on E. coli distribution, reported a greater diversity among 74
pathogenic E. coli populations in horses relative to cattle or humans (Anderson et al. 75
2006). In addition, many of the antimicrobial agents used in the treatment of equine 76
infections belong to the same families as used in human medicine (WHO, 2017). 77
These factors, may contribute to the spread of antibiotic resistance determinants to 78
4
pathogenic strains; as the in vitro and in vivo transmission of resistance markers 79
between species has been described previously (Kelly et al., 2009). 80
Given the high degree of handling and the large number of people (particularly 81
children) who have contact with horses, there is a need to increase awareness of 82
companion/sports animals as potential reservoirs of multi-drug resistant (MDR) and 83
pathogenic E. coli (Sequeria et al., 2009; Murray et al., 2017). The aim of this study 84
was to characterise multidrug resistance determinants and virulence genes and to 85
phenotypically characterise those associated with equine E. coli isolates recovered 86
from foals with diarrhoeal disease. 87
88
Materials and methods 89
Bacterial isolate collection and antimicrobial resistance profiling 90
Faecal samples routinely obtained from foals with enteritis and from post mortem 91
examinations presenting at the Irish Equine Centre in Kildare, Ireland were cultured 92
onto Columbia blood Agar, Wilkins Chalgren agar (anaerobically) and MacConkey 93
agar and incubated overnight at 37°C. Presumptive E. coli were confirmed using API 94
20E strips (bioMériux, Marcy I'Etoile, France) and antibiotic resistance profiles of all 95
isolates were determined against a panel of 14 compounds using disc diffusion, and 96
where appropriate interpreted according to Clinical and Laboratory Standards 97
Institute (CLSI) guidelines (CLSI document VET01-A4, 2013; CLSI supplement 98
VET01S, 2015). Resistance to antimicrobial agents not listed in the CLSI guidelines 99
was determined by the absence of a zone of clearance. The following antimicrobial 100
compounds were included, with their abbreviations and concentrations in 101
parenthesis; ampicillin (A, 10 µg); amikacin (AK, 30 µg); amoxillin/clavulanic acid (AM, 102
20/10 µg); gentamicin (CN, 10 µg); ciprofloxacin (CP, 5 µg); enrofloxacin (E, 5 µg ), 103
5
kanamycin (K, 30 µg), cephalothin (KF, 30 µg), nalidixic acid (NA, 30 µg); norfloxacin 104
(NO, 10 µg); streptomycin (S, 10 µg); sulfonamide (S3, 300 µg); tetracycline (T, 30 105
µg) and trimethoprim (W, 5 µg). All antimicrobial-containing discs were supplied by 106
Oxoid (Fannin Healthcare, Dublin, Ireland). Quality control strains, E. coli 107
ATCC®25922 and Pseudomonas aeruginosa ATCC®27853, were included. Eighty-108
three isolates were identified that were resistant to 3 or more different antimicrobial 109
classes. These were defined as MDR and were subsequently characterised in 110
greater detail, as described below. 111
112
DNA purification 113
Total DNA was prepared from all isolates using the Promega Wizard Genomic DNA 114
purification kit (Madison, WI) following the manufacturer’s instructions. The integrity and 115
concentration of the purified template DNA was assessed by means of conventional agarose 116
gel [1.5%, (w/v)] electrophoresis and by spectrophotometry using a NanoDrop™ ND-1000 117
(Thermoscientific, Wilmington, DE). 118
119
Serotyping 120
Molecular serotyping was performed on all E. coli isolates using PCR amplification of 121
the serogroup specific genes, rfbO26, rfbO111 and rfbO157. 122
A representative group of 25 isolates were then selected at random and sent for 123
conventional serotyping (Public Health England, PHE Colindale, London, UK). 124
125
Pulsed-field gel electrophoresis (PFGE) 126
Molecular subtyping using pulsed-field gel electrophoresis (PFGE) of genomic DNA 127
recovered from equine E. coli isolates was performed according to methods 128
described previously (Duffy et al., 2005). Similarity clustering analyses were 129
6
performed using an unweighted pair group-matching algorithm and the Dice 130
correlation coefficient with a tolerance and optimization of 1.5% with BioNumerics 131
(Applied Maths, Belgium). 132
133
Phylogenetic grouping 134
Phylogenetic groups were determined for each E. coli isolate using an established 135
multiplex PCR targeting chuA, yjaA, and TSPE4.7 according to the protocol of 136
Clermont et al. (2000). Target amplification was performed using the original primer 137
concentrations (Table 1) and cycling conditions (Supplemental Material Table S1). 138
Amplicons generated were separated by conventional 1.7% (w/v) agarose gel 139
electrophoresis, stained with 0.1 g/ml ethidium bromide (Sigma-Aldrich) in 0.5Χ Tris-140
EDTA-boric acid buffer, and subsequently assigned to one of the phylogroups A, B1, 141
B2, or D using the criteria outlined previously by Clermont et al. (2000). 142
143
Adherence and invasion assay 144
Six representative isolates (denoted as Eq23, Eq45, Eq59, Eq67, Eq69 and Eq79) with 145
distinctly different virulence profiles (Supplemental Material Table S2) were selected. These 146
were assessed for their ability to adhere and invade Caco-2 cells as a model for the human 147
small intestinal epithelium. A bacterial adhesion assay was performed with a multiplicity of 148
infection (MOI) of 100 bacteria per epithelial cell according to previously described methods 149
(Simpson et al., 2006). For the invasion assay, Caco-2 monolayers were infected with the 150
same MOI and incubated for 30min at 37°C to allow invasion to occur. The number of 151
intracellular bacteria was determined after the extracellular bacteria were eliminated by 152
incubation of the monolayers with the experimental medium containing gentamicin (100 153
μg/mL) for a further 30min at 37°C. 154
7
All monolayers were subsequently lysed with a 0.5 mL volume of PBS containing 1% 155
(v/v) Triton X-100. Bacteria from each monolayer were collected and plated onto 156
tryptone soy agar (TSA, Sigma) using decimal dilutions. Plate counts from the 157
adhesion assay determined the total number of associated (adhered and invaded) 158
bacteria, and plate counts from the invasion assay determined the number of 159
invaded bacterial cells only. Adherent non-invasive (+-), invasive (++) and non-160
adherent and non-invasive (--) controls were included in each experiment. 161
162
Identification of antimicrobial resistance determinants, integrons, gene 163
cassettes and virulence genes 164
Detection of antibiotic resistance markers and integron-associated genes was 165
performed by PCR, using the primers listed in Table 1. The following resistance 166
determinants were investigated: ampC, blaCTX-M-2, blaOXA, blaSHV, blaTEM, blaPSE, 167
encoding β-lactamases and β-lactamase groups; tet(A) and tet(G) tetracycline efflux 168
pumps; and the sulfonamide resistance gene sul1. 169
A PCR amplification of the gyrA gene was performed on a subset of 16 ciprofloxacin 170
resistant isolates. Previously published PCR methods were employed to determine 171
the presence of conserved integron-associated genes in all isolates, including intI1 172
and intI2 (coding for integrases of classes 1 and 2, respectively), qacEΔ1, sul1, the 173
right-sided conserved segments of class 1 integrons, together with their variable 174
regions (Table 1). Gene cassettes and specific gyrA amplicons of interest were gel 175
extracted using a Qiagen gel extraction kit (West Sussex, UK). DNA was quantified 176
by spectrophotometry and sequenced commercially (Qiagen, Hilden, Germany). 177
Sequence similarity searches were carried out against sequences deposited in the 178
GenBank database using the BLAST search tool 179
8
(https://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence alignments were performed using 180
the online CLUSTALW2 program available at the European Bioinformatics Institute 181
(https://www.ebi.ac.uk/Tools/msa/clustalw2/). 182
All isolates were investigated for the presence of the following virulence-associated 183
genes: stx1 and stx2 which encode Shiga-toxins; eaeA, an adherence factor; the 184
alpha-haemolysin, hlyA; fliCh7, encoding the flagellar antigen H7; cnf1, a cytotoxic 185
necrotizing factor from uropathogenic E. coli; iucD, which encodes the aerobactin 186
operon; afa/draBC, a Dr-binding adhesin (F17 fimbriae); papC, a P fimbriae; 187
sfa/focDE, the S and F1C fimbriae; and neuC, a K1 gene implicated in sialic acid 188
synthesis (Table 1). 189
190
PCR amplification reaction conditions for all of the investigated genes are presented 191
in supplemental material (Supplemental Material Table S1). All reactions contained 192
100 ng of purified DNA, 50 pmol/µL of forward and reverse primers (MWG-Biotech 193
AG, Ebersberg, Germany), 10 X amplification buffer containing 2.5 mM MgCl2, 200 194
µM dNTPs (Promega, Madison, WI) and 0.5 U TaqDNA Polymerase (New England 195
Biolabs, Ipswich, MA) or Pfu Polymerase (Chimerx, Madison, WI). 196
197
Results and Discussion 198
Serotyping 199
Initially, PCR was carried out on all 83 equine E. coli isolates in order to assign them 200
to one of three known pathogenic E. coli serogroups (rfbO157, rfbO26 and rfbO111). One 201
isolate in this study produced a PCR product for serogroup O26, while no amplicons 202
corresponding to the serogroups O157 or O111 were detected (Table 2). However, 203
of the 25 representative isolates sent for conventional serotyping, 10 were typeable 204
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(E. coli O1, O101, O153, O19a, O33, O40, O91; Table 2). All but the O19a 205
serogroup have previously been reported in animal sources including: foals, cattle, 206
pigs, sheep, goats, poultry, cats (Krause et al., 2005; Mora et al., 2011). Serogroups 207
O1, O26, and O101 have previously been associated with both healthy and 208
diarrhoeagenic foals (Holland et al., 1996) however, this is the first reported 209
collection of E. coli isolates from equine sources to contain serogroups O19a, O33, 210
O40, O91 and O153, though they have been previously assessed for biocide 211
tolerance in a separate study (Sheridan et al., 2012). In human infection, all 8 212
serogroups are of clinical significance. All but O19a have previously been associated 213
with human clinical shiga toxin-producing E. coli (STEC) infection (Werberet. al., 214
2008; Vally et al., 2012) and serogroups O1, O19a, O40, O101 and O153 have also 215
been identified among human clinical ExPEC isolates (Ciesielczuk et al., 2016). 216
Novel STEC/ETEC hybrid strains have been recently associated with patients with 217
haemolytic uraemic syndrome (HUS), these included isolates of the serogroups 218
O101 and O153 (Nyholm et al., 2015). The German outbreak involving E. coli 219
reported in 2011 was linked to a hybrid STEC/EAEC, a feature which highlighted the 220
danger of these combinations and the need to routinely screen for multiple sets of 221
virulence factors (Mora et al., 2011). 222
223
224
Virulence gene characterisation 225
Of the 83 equine E. coli isolates examined, none were found to harbour the stx 1 or 226
stx 2 genes; the absence of a single STEC isolate in this collection is unexpected 227
considering the recent increase in outbreaks associated with 228
10
domesticated/companion animals in public settings (Byrne et al., 2015, Murray et al., 229
2017). 230
Of the 11 virulence genes investigated (Table 1), only 4 were detected among these 231
equine E. coli isolates (Table 3). Isolates harbouring two or more of these virulence 232
genes are defined as extraintestinal pathogenic E. coli (ExPEC) (Xia et al., 2011). 233
Thirty-nine (46%) of the isolates in this study were classified as ExPEC on this basis, 234
and hence are considered to be potentially pathogenic to humans and animals. The 235
most commonly detected virulence gene in the collection was iucD (57 %). This gene 236
codes for a siderophore (an iron chelating compound), which enables E. coli to 237
survive in iron-poor environments such as those encountered in UTIs and is related 238
to the virulence of septicemic E. coli from non-equine sources (Siqueria et al., 2009). 239
The occurrence of the fimbrae F17 gene afa/draBC (31%) is comparable to the 240
results of one of few studies available on E. coli in horses (van Duijkeren et al., 241
2000). Van Duijkeren and colleagues identified the presence of afa/draBC in 30% of 242
E. coli recovered from horses with diarrhoeal disease, and none in E. coli from 243
healthy horses, suggesting that these fimbrae might have a role in the cause of 244
diarrhoeal disease in horses. F17 has also been associated with mastitis 245
(Ghanbarpour and Oswald, 2010). The papC gene, producing an adhesin associated 246
with both extraintestinal pathogenicity and an increased capacity to colonize the 247
human intestine, was identified in 36% of isolates. In animals, the papC gene has 248
also been associated with UTIs, respiratory tract infections, soft tissue infections and 249
diarrhoeagenic infection (Siqueria et al., 2009; Ewers et al., 2014). The presence of 250
the sfa/focDE gene was determined in only 2% of isolates. It has been associated 251
with biofilm production in E. coli (Naves et al., 2008); biofilm forming properties are 252
considered important for bacteraemia in the urinary tract of humans and animals 253
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(Wiles et al., 2008). All four virulence determinants are frequently documented in 254
ExPEC E. coli populations and are of clinical significance (Wiles et al., 2008). In 255
addition, we extended the genetic analysis of ten ExPEC isolates by means of a E. 256
coli K12 O157 v2 DNA microarray (Kyle et al. 2010) confirming the presence of 257
these virulence genes and revealing further putative virulence, stress, quorum 258
sensing and antimicrobial resistance (including efflux and porins) genes of interest 259
(Supplemental Material; Word document 1 and Figure S1). These results signal the 260
need for increased attention to be focused on companion/sport animal reservoirs as 261
potential public health risks. 262
263
PFGE subtyping and phylogenetic classification 264
The collection of equine E. coli had a diverse range of pulsotypes of which 33 could 265
be grouped into 9 clusters (C1-9) of 3 or more isolates based on a genetic 266
relatedness criterion of 80% (Figure 1). Six pulsotypes (A, B, C, D, E and J) were 267
common to 2 isolates; 4 pulsotypes (F, G, H, I) were common to 3 isolates; 60 268
isolates had distinct pulsotypes. Pulsotypes A and B were closely related with 269
percentage similarity at 93 % confidence and all 4 isolates belonged to the same 270
phylogenetic group A. 271
272
According to Clermont et al. (2000), E. coli belonging to phylogenetic groups A and 273
B1 are considered non-pathogenic commensal strains, while strains belonging to 274
groups B2 and D are more likely to be pathogenic. The majority of isolates belonged 275
to phylogenetic group A (57 %); while the remainder of isolates belonged to 276
phylogenetic groups B1 (14 %), B2 (6 %) and D (23 %). 277
278
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ExPEC belonging to both pathogenic and non-pathogenic phylogenetic groups were 279
isolated from foals presenting with diarrhoea. This result is in contrast with other 280
studies reporting on equine and other E. coli populations wherein ExPEC isolates 281
predominantly belong to phylogroup B2 (Xia et al., 2011; Ewers et al., 2014). 282
Although typing of human isolates shows good correlation between the phylogenetic 283
group and pathogenicity (Clermont et al., 2000), animal-associated ExPEC can be 284
phylogenetically distinct. Therefore, caution should be exercised when defining such 285
bacteria as commensals (Ghanbarpour and Oswald, 2010). Furthermore, 286
commensal E. coli can cause extra-intestinal disease when predisposing factors for 287
infection are present (Russo and Johnson, 2003). Therefore, although the majority of 288
the isolates investigated in this study may be phylogenetically classified as 289
commensal organisms, they may have the potential to be clinically significant. 290
Further refinement of this phylogenetic classification had recently been documented 291
by Clermont et al. (2013). 292
293
Adherence and invasion 294
Caco-2 cells, a homolog for enterocytes in the intestinal epithelium, were employed 295
to further assess the pathogenicity of isolates with different virulence gene profiles. 296
Bacterial adherence ranged from 6.31 to 7.73 log10cfu mL-1 and bacterial invasion 297
ranged from 1.35 to 5.36 log10cfu mL-1 for all 6 isolates (Figure 2). The non-ExPEC 298
iucD isolate (Eq23) was more adherent to Caco-2 cells (0.85 log10cfu mL-1, p ≤ 299
0.001) compared to the adherent control isolate as well as the other isolates 300
examined. However, this did not result in greater invasive ability. The only isolate to 301
be classed as invasive (5.27 log10cfu mL-1, p ≤ 0.05) was the ExPEC isolate (Eq67) 302
with the iucD-afa/draBC-papC virulence profile. Mellor et al. (2009) demonstrated 303
13
that E. coli isolates considered pathogenic to humans do not display a greater ability 304
to attach to the Caco-2 cell line than those that are not. Perhaps cell lines modelling 305
other common ExPEC sites of infection, such as the human J-82 bladder homolog, 306
might reveal more about the potential these equine ExPEC have to cause infection. 307
308
Antimicrobial resistance profiles 309
All isolates demonstrated an MDR phenotype to critically important antimicrobial 310
agents for human medicine (WHO, 2017). Resistance was demonstrated to between 311
4 and 13 of the antimicrobial agents tested with some 46 different resistance profiles 312
recognised (Supplemental Material Table S2). The majority of isolates (74 %) were 313
resistant to 10 or more antimicrobial compounds. A summary of the frequency of 314
antimicrobial resistance is presented in Table 3.The most common MDR profile 315
among isolates (16 %) had resistance to 12 different compounds, including 316
ampicillin, trimethoprim, ciprofloxacin, streptomycin and tetracycline. Recently, 317
increasing numbers of MDR E. coli have been isolated from animal and human 318
sources (ECDC, 2017; Kennedy et al., 2017) and the acquisition of resistance genes 319
may convey a certain competitive advantage and ultimately lead to increased levels 320
of MDR E. coli in microbial populations (Webber et al., 2017). 321
322
Occurrence of resistance determinants 323
Antimicrobial agents frequently used in veterinary hospitals include broad-spectrum-324
activity drugs, such as β-lactams and fluoroquinolones. Thus, hospitalized animals 325
may constitute an important reservoir of antimicrobial resistance (Karczmarczyk et 326
al., 2011a; WHO, 2017). Twenty-one percent of isolates possessed the commonly 327
reported tet(A) gene, 61% possessed the tet(G) gene and 15% of isolates carried 328
14
both, accounting for 53 of the 81 tetracycline resistant isolates. Sixty-five percent of 329
the MDR equine E. coli isolates harboured one or more TEM, SHV and CTX-M-2 330
group β-lactamases (Table 3), as determined by PCR. The specific variants of these 331
β-lactamases were not identified. AmpC β-lactamases are cephalosporinases that 332
confer resistance to a wide variety of β-lactam drugs and give rise to serious 333
therapeutic challenges in veterinary and human medicine. β-Lactam resistance in E. 334
coli generally occurs as a result of deregulation of the putative ampC gene or the 335
acquisition of a mobile genetic element containing an ampC gene (Li et al., 2007). 336
The presence of plasmid-mediated ampC genes such as the blaCMY variants were 337
not investigated here. In this study, the majority of isolates (79%) were positive for 338
ampC and blaTEM (55%). Isolates with the endogenous ampC gene and the narrow-339
spectrum blaTEM-1 gene are common in animals (Li et al., 2007). Consequently, the 340
high rates of ampicillin-resistant blaTEM-positive isolates among the equine collection, 341
was to be expected (Li et al., 2007). 342
Both TEM and SHV enzymes belong to the class A family of β-lactamases and are 343
widely disseminated among the Enterobacteriaceae from veterinary sources (Li et 344
al., 2007). In this study blaTEM was identified in over half the equine isolates however 345
blaSHV was detected in only 4% of isolates. All were negative for blaPSE or blaOXA. 346
347
TEM enzymes often co-exist with CTX-M enzymes in bacteria of animal origin (Li et 348
al., 2007). CTX-M genes are currently regarded as the predominant extended-349
spectrum β-lactam (ESBL) type of animal origin, while they have also been 350
associated with human isolates in Europe since the late 1990s (Bevan et al., 2017). 351
In Ireland, blaCTX-M-mediated ESBL resistance is widespread (Burke et al., 2016, 352
Morris et al., 2016) however the number of blaCTX-M-2 group-positive isolates in this 353
15
study was low (n=5). To our knowledge, only one instance of blaCTX-M-2 has 354
previously been reported in animals in Ireland, as well as from bacteria of equine 355
origin (Karczmarczyk et al., 2011a). These results are of interest considering the 356
current epidemiology of these genes (Bevan et al., 2017). 357
358
Eighty-six percent of isolates were resistant to nalidixic acid, 79% were resistant to 359
norfloxacin, 77% resistant to enrofloxacin and 73% resistant to ciprofloxacin. The 360
high levels of resistance to quinolones and fluoroquinolones observed in this study 361
are of medical concern, since ciprofloxacin is considered a very valuable 362
antimicrobial agent and is the most effective drug in the treatment of Gram-negative 363
bacterial infections, such as those caused by E. coli and Salmonella species (WHO, 364
2017). Ciprofloxacin resistance is generally conferred by mutations in target genes 365
coding for DNA topoisomerases (gyrA, gyrB, parC, and parE) (Webber et al., 2017). 366
Mutations in the DNA gyrase gyrA have been commonly reported in ciprofloxacin-367
resistant E. coli and Salmonella isolates (Karczmarczyk et al., 2011b). All isolates in 368
this study were found to have two amino acid substitutions in their GyrA subunit 369
(D87Y and S83F). 370
371
Distribution of integrons and gene cassettes 372
Class 1 and class 2 integrons were detected among 85 % of MDR E. coli isolated 373
from diarrhoeagenic foals. Seventy-one percent of isolates were determined to 374
possess the class 1 integrase gene (intI1), and just over half of these (54%) also 375
carried both the qacEΔ1 and sul1 genes. Class 1 integrons may be one of the 376
mechanisms responsible for the rise in MDR E. coli in animal production 377
environments (Kelly et al., 2009; Kennedy et al., 2017). Their contribution to 378
16
resistance appears to be directed against antimicrobial compounds, including 379
streptomycin, trimethoprim and sulfonamides. The presence of class 1 integrons 380
associated with resistance to trimethoprim, streptomycin, and sulfonamide, is in 381
agreement with previous investigations (Kelly et al., 2009; Karczmarczyk et al., 382
2011a; Kennedy et al., 2017). 383
PCR amplification with consensus primers targeting the regions flanking the gene 384
cassettes yielded 6 different amplicons, ranging from 0.5- to 2.5-kbp in size. 385
Integrase I (intI1)-positive isolates possessed none (5%), 1 (31%), 2 (21 %), 3 (5 %), 386
4 (7 %) or 5 (2%) gene cassettes (Supplemental Material Table S2). A 387
representative of each of these 6 gene cassettes was sequenced and annotated. A 388
schematic representation of the gene cassettes is shown in Figure 3. These include 389
the aadA1 and aadA2 genes conferring aminoglycoside resistance, dfrA1 and dfrA2 390
conferring trimethoprim resistance and sat1 conferring streptothricin resistance. 391
Fourteen percent of isolates carried class 2 integrons, as determined by amplification 392
of the integrase gene (intI2). These isolates possessed none (2%), 1 (6%), 2 (4%), 3 393
(1%) or 5 (1%) gene cassettes (Supplemental Material Table S2). The most common 394
cassettes carried by the intI2-positive isolates were the 0.5-kbp (67 %) and the 1.0-395
kbp cassettes (42 %). The variable gene cassette regions from within the integron 396
structures identified were typical of cassettes present in class 1 and class 2 integron 397
structures in E. coli and are widely disseminated among the Enterobacteriaceae 398
(Kadlec et al. 2008). These results reveal the potential to transfer their MDR to other 399
pathogenic and non-pathogenic bacteria. 400
401
The development of MDR in any zoonotic bacterial species gives rise to the potential 402
for it to be transmitted from animals to humans and this could lead to major health 403
17
issues such as the transfer of MDR to other human intestinal mircoflora as well as 404
other human pathogens that are traditionally susceptible to these agents (Kelly et al., 405
2009). MDR infections are associated with poorer clinical outcomes and higher cost 406
of treatment than other infections, and there are already reports of pan-resistant 407
strains in Gram-negative bacteria leading to treatment failure (Xiong et al., 2017). 408
Colonization of diarrhoeagenic foals with MDR E. coli is particularly challenging, 409
given the importance of these drugs. The ongoing usage of antimicrobial compounds 410
in the treatment of animals increases the selective pressure for emergence of MDR 411
organisms and dissemination of resistance (Webber et al., 2017). 412
413
Conclusion 414
The detection of potentially pathogenic MDR equine ExPEC in this study suggests a 415
need for heightened attention to be focused on companion/sport animals as possible 416
sources for human acquisition of disease-causing E. coli. Close monitoring of the 417
virulence and antimicrobial resistance of these bacterial populations, in order to 418
better understand their potential public health risk, is of great importance. 419
420
Acknowledgements 421
Funding for this research was provided by the Irish Equine Centre and through the 422
FIRM fund under the National Development Plan, administered by the Department of 423
Agriculture, Fisheries & Food, Ireland. 424
425
18
References 426
Anderson, M.A., Whitlock, J.E., Harwood, V.J., 2006. Diversity and distribution of 427
Escherichia coli genotypes and antibiotic resistance phenotypes in feces of humans, 428
cattle, and horses. Appl. Environ. Microbiol. 72, 6914-6922. 429
Baucheron, S., Imberechts, H., Chaslus-Dancla, E., Cloeckaert, A., 2002. The AcrB 430
multidrug transporter plays a major role in high-level fluoroquinolone resistance in 431
Salmonella enterica serovar Typhimurium phage type DT204. Microb. Drug 432
Resist. 8, 281-289. 433
Bevan, E.R., Jones, A.M., Hawkey, P.M., 2017. Global epidemiology of CTX-M β-434
lactamases: temporal and geographical shifts in genotype. J. Antimicrob. 435
Chemother. 72, 2145-2155. 436
Burke, L., Humphreys, H. and Fitzgerald-Hughes, D., 2016. The Molecular 437
Epidemiology of Resistance in Cefotaximase-Producing Escherichia coli Clinical 438
Isolates from Dublin, Ireland. Microb. Drug Resist. 22, 552-558. 439
Byrne, L., Jenkins, C., Launders, N., Elson, R., Adak, G., 2015. The epidemiology, 440
microbiology and clinical impact of Shiga toxin-producing Escherichia coli in 441
England, 2009–2012. Epidemiol. Infect. 143, 3475-3487. 442
Ciesielczuk, H., Jenkins, C., Chattaway, M., Doumith, M., Hope, R., Woodford, N., 443
Wareham, D.W., 2016. Trends in ExPEC serogroups in the UK and their 444
significance. Clin. Microbiol. Infect. 35, 1661-1666. 445
Clermont, O., Bonacorsi, S., Bingen, E., 2000.Rapid and simple determination of the 446
Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66, 4555-4558. 447
Clermont, O., Christenson, J.K., Denamur, E., Gordon, D.M., 2013. The Clermont 448
Escherichia coli phylotyping method revisited: improvement of specificity and 449
detection of new phylogroups. Environ. Microbiol. Rep. 5, 58-65. 450
19
Clinical and Laboratory Standards Institute (CLSI) 2013. Performance standards for 451
antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 452
4th ed. CLSI document VET01-A4. Clinical and Laboratory Standards Institute, 950 453
West Valley Road, Suite 2500, Wayne, PA 19087 USA. 454
Clinical and Laboratory Standards Institute (CLSI) 2015. Performance standards for 455
antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 456
3rd ed. CLSI supplement VET01S. Clinical and Laboratory Standards Institute, 950 457
West Valley Road, Suite 2500, Wayne, PA 19087 USA. 458
Croxen, M.A., Law, R.J., Scholz, R., Keeney, K.M., Wlodarska, M., Finlay, B.B., 459
2013. Recent advances in understanding enteric pathogenic Escherichia coli. Clin. 460
Microbiol. Rev. 26, 822-880. 461
DebRoy, C., Roberts, E., Kundrat, J., Davis, M.A., Briggs, C.E., Fratamico, P.M., 462
2004. Detection of Escherichia coli serogroups O26 and O113 by PCR amplification 463
of the wzx and wzy genes. Appl. Environ. Microbiol. 70, 1830-1832. 464
Duffy, G., O'Brien, S.B., Carney, E., Sheridan, J.J., McDowell, D.A., Blair, I.S., 2005. 465
Characterisation of E. coli O157 isolates from bovine hide and beef trimming in Irish 466
abattoirs by pulsed field gel electrophoresis. J. Microbiol. Methods 60, 375-382. 467
European Centre for Disease Prevention and Control.Surveillance of antimicrobial 468
resistance in Europe 2016.Annual Report of the European Antimicrobial Resistance 469
Surveillance Network (EARS-Net). Stockholm: ECDC; 2017. 470
Ewers, C., Bethe, A., Stamm, I., Grobbel, M., Kopp, P.A., Guerra, B., Stubbe, M., 471
Doi, Y., Zong, Z., Kola, A., Schaufler, K., 2014. CTX-M-15-D-ST648 Escherichia coli 472
from companion animals and horses: another pandemic clone combining 473
multiresistance and extraintestinal virulence?. J. Antimicrob. Chemother. 69, 1224-474
1230. 475
20
Gannon, V.P., King, R.K., Kim, J.Y., Thomas, E.J., 1992. Rapid and sensitive 476
method for detection of Shiga-like toxin-producing Escherichia coli in ground beef 477
using the polymerase chain reaction. Appl. Environ. Microbiol. 58, 3809-3815. 478
Ghanbarpour, R., Oswald, E., 2010. Phylogenetic distribution of virulence genes in 479
Escherichia coli isolated from bovine mastitis in Iran. Res. Vet. Sci. 88, 6-10. 480
Holland, R.E., Schmidt, A., Sriranganathan, N., Grimes, S.D., Wilson, R.A., Brown, 481
C.M., Walker, R.D., 1996. Characterization of Escherichia coli isolated from foals. 482
Vet. Microbiol. 48, 243-255. 483
Johnson, J.R., Stell, A.L., 2000. Extended virulence genotypes of Escherichia coli 484
strains from patients with urosepsis in relation to phylogeny and host compromise. J. 485
Infect. Dis. 181, 261-272. 486
Kadlec, K., Schwarz, S., 2008. Analysis and distribution of class 1 and class 2 487
integrons and associated gene cassettes among Escherichia coli isolates from 488
swine, horses, cats and dogs collected in the BfT-GermVet monitoring study. J. 489
Antimicrob. Chemother. 62, 469-473. 490
Karczmarczyk, M., Abbott, Y., Walsh, C., Leonard, N. and Fanning, S., 2011a. 491
Characterization of multidrug-resistant Escherichia coli isolates from animals 492
presenting at a university veterinary hospital. Appl. Environ. Microbiol. 77, 7104-493
7112. 494
Karczmarczyk, M., Martins, M., Quinn, T., Leonard, N. and Fanning, S., 495
2011b.Mechanisms of fluoroquinolone resistance in Escherichia coli isolates from 496
food-producing animals. Appl. Environ. Microbiol. 77, 7113-7120. 497
Kelly, B.G., Vespermann, A., Bolton, D.J., 2009. Horizontal gene transfer of virulence 498
determinants in selected bacterial foodborne pathogens. Food Chem. Toxicol. 47, 499
969-977. 500
21
Kennedy, C.A., Fanning, S., Karczmarczyk, M., Byrne, B., Monaghan, A., Bolton, D., 501
Sweeney, T., 2017. Characterizing the multidrug resistance of non-O157 Shiga 502
Toxin-Producing Escherichia coli isolates from cattle farms and abattoirs. Microb. 503
Drug Resist. 23, 781-790. 504
Krause, G., Zimmermann, S., Beutin, L., 2005. Investigation of domestic animals and 505
pets as a reservoir for intimin-(eae) gene positive Escherichia coli types. Vet. 506
Microbiol. 106, 87-95. 507
Kyle, J.L., Parker, C.T., Goudeau, D., Brandl, M.T., 2010.Transcriptome analysis of 508
Escherichia coli O157:H7 exposed to lysates of lettuce leaves. Appl. Environ. 509
Microbiol. 76, 1375-87. 510
Lenahan, M., O’Brien, S., Kinsella, K., Sweeney, T., Sheridan, J.J., 2007. 511
Prevalence and molecular characterization of Escherichia coli O157: H7 on Irish 512
lamb carcasses, fleece and in faeces samples. J. Appl. Microbiol. 103, 2401-2409. 513
Lenahan, M., O’Brien, S.B., Byrne, C., Ryan, M., Kennedy, C.A., McNamara, E.B., 514
Fanning, S., Sheridan, J.J., Sweeney, T., 2009. Molecular characterization of Irish E. 515
coli O157: H7 isolates of human, bovine, ovine and porcine origin. J. Appl. Microbiol. 516
107, 1340-1349. 517
Li, X.Z., Mehrotra, M., Ghimire, S., Adewoye, L., 2007.β-Lactam resistance and β-518
lactamases in bacteria of animal origin. Vet. Microbiol. 121, 197-214. 519
Mellor, G.E., Goulter, R.M., Chia, T.R., Dykes, G.A., 2009. Comparative analysis of 520
attachment of Shiga-toxigenic Escherichia coli and Salmonella strains to cultured 521
HT-29 and Caco-2 cell lines. Appl. Environ. Microbiol. 75, 1796-1799. 522
Mora, A., Herrrera, A., López, C., Dahbi, G., Mamani, R., Pita, J.M., Alonso, M.P., 523
Llovo, J., Bernárdez, M.I., Blanco, J.E., Blanco, M., 2011. Characteristics of the 524
22
Shiga-toxin-producing enteroaggregative Escherichia coli O104: H4 German 525
outbreak strain and of STEC strains isolated in Spain. Int. Microbiol. 14, 121-141. 526
Morris, D., O'connor, M., Izdebski, R., Corcoran, M., Ludden, C.E., McGrath, E., 527
Buckley, V., Cryan, B., Gniadkowski, M., Cormican, M., 2016. Dissemination of 528
clonally related multidrug-resistant Klebsiella pneumoniae in Ireland. Epidemiol. 529
Infect. 144, 443-448. 530
Murray, R., Tataryn, J., Pintar, K., Thomas, M.K., 2017. Estimates of the burden of 531
illness for eight enteric pathogens associated with animal contact in Canada. 532
Epidemiol. Infect. 145, 3413-3423. 533
Naves, P., del Prado, G., Huelves, L., Gracia, M., Ruiz, V., Blanco, J., Dahbi, G., 534
Blanco, M., del Carmen Ponte, M., Soriano, F., 2008. Correlation between virulence 535
factors and in vitro biofilm formation by Escherichia coli strains. Microb Pathog 45, 536
86-91. 537
Nyholm, O., Heinikainen, S., Pelkonen, S., Hallanvuo, S., Haukka, K., Siitonen, A., 538
2015. Hybrids of Shigatoxigenic and enterotoxigenic Escherichia coli (STEC/ETEC) 539
among human and animal isolates in Finland. Zoonoses Public Health 62, 518-524. 540
Paton, A.W., Paton, J.C., 1998. Detection and characterization of Shiga toxigenic 541
Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, 542
enterohemorrhagic E. coli hlyA, rfb O111, and rfb O157. J. Clin. Microbiol. 36, 598-543
602. 544
Russo, T.A., Johnson, J.R., 2003. Medical and economic impact of extraintestinal 545
infections due to Escherichia coli: focus on an increasingly important endemic 546
problem. Microb. Infect. 5, 449-456. 547
23
Sandvang, D., Aarestrup, F.M., Jensen, L.B., 1998. Characterisation of integrons 548
and antibiotic resistance genes in Danish multiresistant Salmonella 549
entericaTyphimurium DT104. FEMS Microbiol. Lett. 160, 37-41. 550
Sheridan, À., Lenahan, M., Duffy, G., Fanning, S., Burgess, C., 2012. The potential 551
for biocide tolerance in Escherichia coli and its impact on the response to food 552
processing stresses.Food Control 26, 98-106. 553
Siqueira, A.K., Ribeiro, M.G., Leite, D.D.S., Tiba, M.R., de Moura, C., Lopes, M.D., 554
Prestes, N.C., Salerno, T., da Silva, A.V., 2009. Virulence factors in Escherichia coli 555
strains isolated from urinary tract infection and pyometra cases and from feces of 556
healthy dogs. Res. Vet. Sci. 86, 206-210. 557
Simpson, K.W., Dogan, B., Rishniw, M., Goldstein, R.E., Klaessig, S., McDonough, 558
P.L., German, A.J., Yates, R.M., Russell, D.G., Johnson, S.E., Berg, D.E., 2006. 559
Adherent and invasive Escherichia coli is associated with granulomatous colitis in 560
boxer dogs. Infect. Immun. 74, 4778-4792. 561
Vally, H., Hall, G., Dyda, A., Raupach, J., Knope, K., Combs, B., Desmarchelier, P., 562
2012. Epidemiology of Shiga toxin producing Escherichia coli in Australia, 2000-563
2010.BMC Public Health 12, 63-74. 564
Van Duijkeren, E., Van Asten, A.J.A.M., Gaastra, W., 2000. Characterization of 565
Escherichia coli isolated from adult horses with and without enteritis. Vet. Quart. 22, 566
162-166. 567
Wiles, T.J., Kulesus, R.R., Mulvey, M.A., 2008. Origins and virulence mechanisms of 568
uropathogenic Escherichia coli. Exp. Mol. Pathol. 85, 11-19. 569
Winokur, P.L., Brueggemann, A., DeSalvo, D.L., Hoffmann, L., Apley, M.D., 570
Uhlenhopp, E.K., Pfaller, M.A., Doern, G.V., 2000. Animal and human multidrug-571
resistant, cephalosporin-resistant Salmonella isolates expressing a plasmid-572
24
mediated CMY-2 AmpC β-lactamase. Antimicrob. Agents Chemother. 44, 2777-573
2783. 574
Webber, M.A., Buckner, M.M., Redgrave, L.S., Ifill, G., Mitchenall, L.A., Webb, C., 575
Iddles, R., Maxwell, A., Piddock, L.J., 2017. Quinolone-resistant gyrase mutants 576
demonstrate decreased susceptibility to triclosan. J. Antimicrob. Chemother. 72, 577
2755-2763. 578
Werber, D., Beutin, L., Pichner, R., Stark, K., Fruth, A., 2008. Shiga toxin–producing 579
Escherichia coli serogroups in food and patients, Germany. Emerg. Infect. Diseases 580
14, 1803-1806. 581
World Health Organization, 2017. Critically important antimicrobials for human 582
medicine: ranking of antimicrobial agents for risk management of antimicrobial 583
resistance due to non-human use. ISBN 978-92-4-151222-0 584
Xia, X., Meng, J., Zhao, S., Bodeis-Jones, S., Gaines, S.A., Ayers, S.L., McDermott, 585
P.F., 2011. Identification and antimicrobial resistance of extraintestinal pathogenic 586
Escherichia coli from retail meats. J. Food Prot. 74, 38-44. 587
Xiong, J., Déraspe, M., Iqbal, N., Krajden, S., Chapman, W., Dewar, K., Roy, P.H., 588
2017. Complete genome of a panresistant Pseudomonas aeruginosa strain, isolated 589
from a patient with respiratory failure in a canadian community hospital. Genome 590
Announc. 5, e00458-17. 591
Xu, L., Ensor, V., Gossain, S., Nye, K., Hawkey, P., 2005. Rapid and simple 592
detection of blaCTX−M genes by multiplex PCR assay. J. Med. Microbiol. 54, 1183-593
1187. 594
Zhou, X.Y., Bordon, F., Sirot, D., Kitzis, M.D., Gutmann, L., 1994.Emergence of 595
clinical isolates of Escherichia coli producing TEM-1 derivatives or an OXA-1 beta-596
25
lactamase conferring resistance to beta-lactamase inhibitors. Antimicrob. Agents 597
Chemother. 38, 1085-1089. 598
599
Tables 600
Table 1: PCR primer characteristics 601
Table 2: Typable equine E. coli strains as determined by Public Health England 602
Table 3: The percentage of antibiotic resistance, resistance markers and virulence 603
genes among equine E. coli isolates. 604
605
Figure captions 606
Figure 1: Dendrogram showing genotypic similarities between the equine E. coli 607
isolates (n=83) based on pulsotypes. 608
Figure 2: Adherence (A) and invasion (B) of Caco-2 cells by equine E. coli isolates 609
positive or negative for virulence genes. 610
Figure 3: Schematic representation of the organization of sequenced gene cassettes 611
of class 1 integrons. 612
613
1
Figure legends 1
2
Figure 2 legend: Adhesion (A) and invasion (B) of equine E. coli to human Caco-2 3
cells. Values indicate means of three separate experiments ± S.E. The results of 4
statistical analysis are indicated by the letters a, b and c. Strains with different letters 5
are significantly different from each other (p ˂ 0.05). 6
Figure 3 legend: aad - aminoglycoside adenyltransferase; attl1 - Integron associated 7
recombination site; dfr - dihydrofolate reductase; sat1 - streptothricin acetyl 8
transferase; 59bp - 59 base pair element recombination site; hp - hypothetical 9
protein; CS - conserved segment. Arrowheads represent the direction of the 10
transcription. 11
1
Table 1:PCR primer characteristics 1
Sensea Primer sequence (5’-to-3’) Reference
Serogroup rfbO157
F R
CGG ACA TCC ATG TGA TAT GG TTG CCT ATG TAC AGC TAA TCC
Paton and Paton, 1998
rfbO111
F R
TAG AGA AAT TAT CAA GTT AGT TCC ATA GTT ATG AAC ATC TTG TTT AGC
Paton and Paton, 1998
rfbO26
F R
GCG CTG CAA TTG CTT ATG TA TTT CCC CGC AAT TTA TTC AG
Debroy et al., 2004
Phylogenetic chuA F GACGAACCAACGGTCAGGAT Clermont et al., 2000 Group R TGCCGCCAGTACCAAAGACA
yjaA F TGAAGTGTCAGGAGACGCTG Clermont et al., 2000
R ATGGAGAATGCGTTCCTCAAC
TSPE4C2 F GAGTAATGTCGGGGCATTCA Clermont et al., 2000
R CGCGCCAACAAAGTATTGCG Virulence Gene
stx1
F R
ACA CTG GAT GAT CTC AGT GG CTG AAT CCC CCT CCA TTA TG
Gannon et al., 1992
stx2
F R
CCA TGA CAG ACG GAC AGC AGT T CCT GTC AAG CTG AGC ACT TTG
Gannon et al., 1992
eaeA
F R
GAC CCG GCA CAA GCA TAA GC CCA CCT GCA GCA ACA AGA GG
Lenahan et al., 2007
hlyA
F R
GCA TCA TCA AGC GTA CGT TCC AAT GAG CCA AGC TGG TTA AGC T
Lenahan et al., 2007
fliCh7
F R
GCGCTGTCGAGTTCTATCGAGC CAACGGTGACTTTATCGCCATTCC
Lenahan et al., 2007
cnf1
F R
GAA CTT ATT AAG GAT AGT CAT TAT TTA TAA CGC TG
Siqueira et al., 2009
iucD (aerobactin operon)
F R
TAC CGG ATT GTC ATA TGC AGA CCG T AAT ATC TTC CTC CAG TCC GGA GAA G
Siqueira et al., 2009
papC
F R
GAC GGC TGT ACT GCA GGG TGT GGC G ATA TCC TTT CTG CAG GGA TGC AAT A
Siqueira et al., 2009
sfa/focDE
F R
CTC CGG AGA ACT GGG TGC ATC TTA C CGG AGG AGT AAT TAC AAA CCT GGC A
Siqueira et al., 2009
afa/draBC
F R
GCT GGG CAG CAA ACT GAT AAC TCT C CAT CAA GCT GTT TGT TCG TCC GCC G
Siqueira et al., 2009
neuC(K1)
F R
AGG TGA AAA GCC TGG TAG TGT G GGT GGT ACA TCC CGG GAT GTC
Johnson & Stell, 2000
AR Gene blaTEM
F R
GTA TGG ATC CTC AAC ATT TCC GTG TCG ACC AAA GCT TAA TCA GTG AGG CA
Zhou et al., 1994
blaPSE
F R
CGC TTC CCG TTA ACA ACT AC CTG GTT CAT TTC AGA TAG CG
Winokur et al., 2000
blaSHV
F R
TCA GCG AAA AAC ACC TTG TCC CGC AGA TAA ATC ACC A
Kennedy et al., 2017
blaOXA
F R
TAT CTA CAG CAG CGC CAG TG CGC ATC AAA TGC CAT AAG TG
Kennedy et al., 2017
ampC
F R
CCC CGC TTA TAG AGC AAC AA TCA ATG GTC GAC TTC ACA CC
Kennedy et al., 2017
tet(A)
F R
GCT ACA TCC TGC TTG CCT TC CAT AGA TCG CCG TGA AGA GG
Kennedy et al., 2017
tet(G)
F R
GCT CGG TGG TAT CTC TGC TC AGC AAC AGA ATC GGG AAC AC
Kennedy et al., 2017
blaCTX-M-2 group
F R
TGA TAC CAC CAC GCC GCT C TAT TGC ATC AGA AAC CGT GGG
Xu et al., 2005
gyrA
F R
TGT CCG AGA TGG CCT GAA GC CGT TGA TGA CTT CCG TCA G
Baucheron et al., 2002
Integron Components
gene cassette F R
GGC ATC CAA GCA GCA AGC AAG CAG ACT TGA CCT GAT
Sandvang et al., 1998
intI1
F R
GTG GAT GGC GGC CTG AAG CC ATT GCC CAG TCG GCA GCG
Sandvang et al., 1998
intI2
F R
CAC GGA TAT GCG ACA AAA AGG T GTA GCA AAC GAG TGA CGA AAT G
Kennedy et al., 2017
sul1
F R
CTT CGA TGA GAG CCG GCG GC GCA AGG CGG AAA CCC GCG CC
Sandvang et al., 1998
qacE∆1
F R
ATC GCA ATA GTT GGC GAA GT CAA GCT TTT GCC CAT GAA GC
Sandvang et al., 1998
aF, forward; R, reverse; AR, antibiotic resistance target 2
1
Table 2: Typeable E. coli strains as determined by conventional serotyping of 25 1
representative E. coli isolates and in-house PCR recovered from diarrhoeagenic 2
foals by the Irish Equine Centre 3
Isolate Code Serotype
Conventional Serotyping Eq4 E. coli O1
Eq48 E. coli O1
Eq74 E. coli O101
Eq50 E. coli O153
Eq77 E. coli O153
Eq44 E. coli O19a
Eq54 E. coli O33
Eq45 E. coli O40
Eq75 E. coli O91
In house PCR serotyping Eq64 E. coli O26
Serotyping results for the remaining 16 isolates were not available, as the isolates were 4
‘unidentifiable’ using traditional serotyping techniques. 5
1
Table 3: Antibiotic resistance, associated resistance markers, and virulence genes 1
among equine E. coli isolates 2
Antimicrobial Agents % Resistance
Ampicillin* 93
Amikacin* 6
Amoxillin/clavulanic acid* 30
Gentamicin* 68
Ciprofloxacin* 73
Enrofloxacin* 77
Kanamycin* 70
Cefalothin 67
Naladixic Acid* 86
Norfloxacin* 79
Streptomycin* 98
Sulfonamide 98
Tetracycline 96
Trimethoprim 98
Integron Components % Present
intI1 71
intI2 14
qacE∆1 71
sul1 57
Gene Cassettes (kb) % Present
0.5 64
1.0 40
1.5 35
1.7 10
2.0 4
2.5 6
Antibiotic Resistance Determinants
% Present
ampC 80
blaSHV 4
tet(A) 21
tet(G) 61
blaTEM 55
blaCTX-M-2 group 6
Virulence Genes % Present
iucD 57
afa/draBC 31
papC 36
sfa/focDE 2
*Critically important antimicrobial agents (WHO, 2017)3
2